Nontypable Haemophilus influenzae (NTHi) is a major cause of acute otitis media (AOM) accounting for 20-30% of all episodes. There is also evidence that NTHi is the most frequent pathogen in children with recurrent episodes of AOM. The extensive heterogeneity of outer membrane proteins has been an impediment to identifying effective vaccine candidates against NTHi. In animal models, membrane protein antigens tend to induce homologous, but not heterologous protection. To date, the PI's research on H. influenzae has exclusively focused on phylogenetic characterization of species natural population structure and its use to survey degree of conservation of potential outer membrane protein targets. Because few essential virulence factors have been identified in the pathogenesis of AOM caused by NTHi, the PI carried out a preliminary pilot study to test the hypothesis that sialic acid, a terminal sugar of NTHi lipopolysaccharicle, might be an essential virulence factor in AOM, analogous to the virulence role of sialic acid of LPS in gonococcal and meningococcal infections. These studies capitalized upon the availability of: (i) isogenic pairs of wild-type and LPS sialylation deficient mutants; (ii) the chinchilla animal model for experimental otitis media and (iii) the fine structural analysis of LPS, including data on organisms obtained ex-vivo from the animal model. This pilot study revealed attenuation of infection by the sialic acid deficient mutant strains compared to infection with wild-type organisms. This is consistent with prior studies showing that sialic acid deficient mutants of NTHi are relatively susceptible to complement mediated bactericidal killing by pooled human sera. The current R21 proposal aims to expand this preliminary evaluation of the role of sialic acid in the pathogenesis of AOM. It amalgamates population biology, a validated animal model of AOM, microbial genetics, and structural analysis to determine the role of sialic acid as virulence factor. These data will pave the way for further studies that could be supported by a future R01 application.